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2007 l-Fucose Stimulates Utilization of d- by Escherichia coli MG1655 ΔfucAO and E. coli Nissle 1917 ΔfucAO Mutants in the Mouse Intestine and in M9 Minimal Medium Steven M. Autieri University of Rhode Island

Jeremy J. Lins University of Rhode Island

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Citation/Publisher Attribution Autieri, S. M., Lin, J. J., Leatham, M. P., Laux, D. C., Conway, T., & Cohen, P. S. (2007). l-Fucose Stimulates Utilization of d-Ribose by Escherichia coli MG1655 ΔfucAO and E. coli Nissle 1917 ΔfucAO Mutants in the Mouse Intestine and in M9 Minimal Medium. Infection and Immunity, 75(11), 5465-5475. doi: 10.1128/IAI.00822-07 Available at: http://dx.doi.org/10.1128/IAI.00822-07

This Article is brought to you for free and open access by the Cell and Molecular Biology at DigitalCommons@URI. It has been accepted for inclusion in Cell and Molecular Biology Faculty Publications by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. Authors Steven M. Autieri, Jeremy J. Lins, Mary P. Leatham, David C. Laux, Tyrrell Conway, and Paul S. Cohen

This article is available at DigitalCommons@URI: https://digitalcommons.uri.edu/cmb_facpubs/41 INFECTION AND IMMUNITY, Nov. 2007, p. 5465–5475 Vol. 75, No. 11 0019-9567/07/$08.00ϩ0 doi:10.1128/IAI.00822-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

L-Fucose Stimulates Utilization of D-Ribose by Escherichia coli MG1655 ⌬fucAO and E. coli Nissle 1917 ⌬fucAO Mutants in the Mouse Intestine and in M9 Minimal Mediumᰔ Steven M. Autieri,1 Jeremy J. Lins,1 Mary P. Leatham,1 David C. Laux,1 Tyrrell Conway,2 and Paul S. Cohen1* Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881,1 and Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 730192

Received 15 June 2007/Returned for modification 30 July 2007/Accepted 7 August 2007 Downloaded from

Escherichia coli MG1655 uses several for growth in the mouse intestine. To determine the roles of L-fucose and D-ribose, an E. coli MG1655 ⌬fucAO mutant and an E. coli MG1655 ⌬rbsK mutant were fed separately to mice along with wild-type E. coli MG1655. The E. coli MG1655 ⌬fucAO mutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but the E. coli MG1655 ⌬rbsK mutant and the wild type colonized at nearly identical levels. Surprisingly, an E. coli MG1655 ⌬fucAO ⌬rbsK mutant was eliminated from the intestine by either wild-type E. coli MG1655 or E. coli MG1655 ⌬fucAO, suggesting that the ⌬fucAO mutant switches to ribose in vivo. Indeed, in vitro growth experiments showed that L-fucose stimulated utilization of D-ribose by the E. coli MG1655 ⌬fucAO mutant but not by an E. coli MG1655 ⌬fucK

mutant. Since the ⌬fucK mutant cannot convert L- to L-fuculose-1-phosphate, whereas the ⌬fucAO http://iai.asm.org/ mutant accumulates L-fuculose-1-phosphate, the data suggest that L-fuculose-1-phosphate stimulates growth on ribose both in the intestine and in vitro. An E. coli Nissle 1917 ⌬fucAO mutant, derived from a human probiotic commensal strain, acted in a manner identical to that of E. coli MG1655 ⌬fucAO in vivo and in vitro. Furthermore, L-fucose at a concentration too low to support growth stimulated the utilization of ribose by the wild-type E. coli strains in vitro. Collectively, the data suggest that L-fuculose-1-phosphate plays a role in the regulation of ribose usage as a carbon source by E. coli MG1655 and E. coli Nissle 1917 in the mouse intestine.

We are interested in identifying the nutrients that Esche- make up the microflora (39) can coexist in the large intestine on October 11, 2018 by guest richia coli uses to successfully colonize the mouse large intes- as long as each member is able to utilize one or a few limiting tine in the face of competition from an extensive microflora. nutrients better than all the others and that the rate of growth The large intestine of the mouse consists of the cecum and the of any member of the microflora that successfully colonizes is colon, each of which contains the mucosa and the luminal at least equal to the washout rate from the intestine (19, 20, contents. The two components of the mucosa are the layer of 21). It is the presence of a complete microflora that results in epithelial cells on the intestinal wall and the mucus layer that what has been termed colonization resistance, which refers to covers them. The relatively thick (up to 800 ␮m) mucus layer the ability of the intestinal microflora to resist colonization by consists of mucin, a 2-MDa gel-forming and a an invading bacterium (2, 20, 22, 54). Due to colonization large number of smaller , proteins, glycolipids, resistance, studies aimed at determining how an invading bac- lipids, and sugars (1, 3, 15, 28, 46, 47, 51, 52). The mucus layer terium colonizes the mouse intestine are difficult, if not impos- itself is in a dynamic state, constantly being synthesized and sible, with conventional animals. However, intestinal coloniza- secreted by specialized goblet cells and degraded to a large tion can be studied with the streptomycin-treated mouse, extent by the indigenous intestinal microbes (25, 41). E. coli is which has been used extensively to study E. coli, Salmonella present both in mucus and luminal contents; however, a large enterica serovar Typhimurium, and Klebsiella pneumoniae in- body of experimental evidence shows that growth is rapid in testinal colonization (14, 34, 42, 43). intestinal mucus both in vitro and in vivo but is either poor or Streptomycin treatment alters the microecology of the ce- completely inhibited in luminal contents (33, 38, 43, 52, 53, 55). cum, decreasing the populations of facultative anaerobes and It is therefore highly likely that the ability of an E. coli strain to some strict anaerobes and creating a niche for organisms such grow in intestinal mucus plays a critical role in its ability to as E. coli (23). Nevertheless, populations of the genera Bacte- colonize the intestine. roides and Eubacterium in cecal contents of streptomycin- The prevalent theory as to how bacteria colonize the mam- treated mice remain largely unchanged (23). Moreover, the malian gut has been called the nutrient-niche theory. This overall number of strict anaerobes in the cecal contents of theory states that the 500 to 1,000 indigenous species that streptomycin-treated and conventional mice is essentially iden- tical (1 ϫ 109 to 2 ϫ 109 CFU/g of contents) (23). Therefore, while the streptomycin-treated mouse model is not perfect, * Corresponding author. Mailing address: Department of Cell and invading microorganisms must compete for nutrients with a Molecular Biology, University of Rhode Island, Kingston, RI 02881. Phone: (401) 874-5920. Fax: (401) 874-2202. E-mail: pco1697u large number of strict anaerobes in the intestine, just as they do @postoffice.uri.edu. in conventional animals. This feature makes the streptomycin- ᰔ Published ahead of print on 20 August 2007. treated mouse model the one of choice to identify the nutrients

5465 5466 AUTIERI ET AL. INFECT.IMMUN.

E. coli uses to colonize the mouse intestine in the face of monitored spectrophotometrically (A600) using a Pharmacia Biotech Ultrospec competition from an extensive microflora. 2000 UV/visible spectrophotometer. Mutant construction. E. coli Using the streptomycin-treated mouse model, it recently has Mutant strains were created by deletion mu- tagenesis using either a chloramphenicol cassette or a kanamycin cassette as been shown that E. coli MG1655, a human commensal strain described by Datsenko and Wanner (12). Primers used to construct deletion for which the genome has been completely sequenced (5), mutants were designed according to the E. coli MG1655 genome sequence utilizes several sugars simultaneously for growth in the mouse (5). DNA procedures were as described previously (37). All constructs were verified ⌬ intestine (7). While these results support the nutrient-niche by PCR and sequencing. The primers used to construct the fucAO deletions were the following (uppercase letters, MG1655 DNA; lowercase letters, kanamycin cas- theory of colonization, the mechanisms governing the simulta- sette DNA): primer 1, 5Ј-CGCAAATGTGGCGGAATACCGACATCACGGTT neous use of sugars are largely unknown. In the present study, GAGAGCAAACAgtgtaggctggagctgcttcg-3Ј; primer 2, 5Ј-CGTCAGTGTACGTTA we report that while studying the role of L-fucose as a nutrient TCAGGATGGGATGCTGATTACGCCTACAGGCAtatgaatatcctccttag-3Ј. The for E. coli MG1655 and E. coli Nissle 1917 in the mouse primers used to construct the ⌬rbsK deletions were the following (uppercase letters, MG1655 DNA; lowercase letters, chloramphenicol or kanamycin cassette DNA): intestine, we found that accumulation of L-fuculose-1-phos- primer 1, 5Ј-TGGCAGCATTAATGCTGACCACATTCTTAATCTTCAATCTT phate, an intermediate in the degradation of L-fucose in E. coli, TTCCTACTCtgtaggctggagctgcttcg-3Ј; primer 2, 5Ј-GGTACGGAAGGTTGTGCG

stimulates utilization of D-ribose. The present studies therefore CCTTTACGTGTTACGGCAATCGCAGCGGCAGGCAtatgaatatcctccttag-3Ј. The Downloaded from raise the possibility that pool sizes of metabolic intermediates primers used to construct the ⌬fucK deletions were the following (uppercase play a role in the selection of nutrients used by E. coli MG1655 letters, MG1655 DNA; lowercase letters, chloramphenicol or kanamycin cassette DNA): primer 1, 5Ј-CTGTGGCGCGACCAATGTCAGGGCCATCGCGGTT and E. coli Nissle 1917 to achieve maximum colonizing ability. AATCGGCAGGGgtgtaggctggagctgcttcg-3Ј; primer 2, 5Ј-GTACGACTTAACA GCGAAGTATCAACCTGGGCGCTGCGAACCATCAtatgaatatcctccttag-3Ј. Sequencing. DNA sequencing was done at the URI Genomics and Sequencing MATERIALS AND METHODS Center, University of Rhode Island, Kingston, using the CEQ8000 genetic anal- Bacterial strains. E. coli MG1655 Strr is a spontaneous streptomycin-resistant ysis system (Beckman Coulter, Fullerton, CA). The dye terminator cycle se- mutant of the sequenced wild-type E. coli MG1655 strain (CGSC 7740) (37). E. quencing quick start kit (Beckman Coulter) was used in the sequencing reactions. coli MG1655 Strr Nalr is a spontaneous nalidixic acid-resistant mutant of E. coli The primers used to amplify PCR products for sequencing to determine the ⌬ http://iai.asm.org/ MG1655 Strr (37). It is referred to in the text as E. coli MG1655. The following precise location of the deletions for E. coli MG1655 fucK and E. coli Nissle 1917 ⌬ Ј Ј Ј deletion mutants were constructed from E. coli MG1655 Strr as described below fucK were 5 -TATGCACAACGTTGAAGACACC-3 and 5 -CCACAATGT Ј in the section on mutant construction. E. coli MG1655 Strr ⌬fucAO::kan is both GTTGCGACTTCCTC-3 . The same primers were used for sequencing. For E. ⌬ ⌬ streptomycin resistant and kanamycin resistant and has a 1,542-bp deletion coli MG1655 fucAO and E. coli Nissle 1917 fucAO PCR amplification, the Ј Ј Ј within the 1,823-bp fucA and fucO genes. It is unable to convert fuculose-1- primers were the following: 5 -GCTTACAAACCGATTTGCATATC-3 and 5 - Ј Ј phosphate to phosphate and lactaldehyde and lacks both L- GTGGGTAATTAAACGGCTAATTC-3 ; for sequencing, the primers were 5 - Ј Ј fuculose phosphate aldolase and propanediol oxidoreductase. It is referred to in AACAGTACTGCGATGAGTGGCAG-3 and 5 -GCGAAGTGATCTTCCGT Ј ⌬ the text as E. coli MG1655 ⌬fucAO. E. coli MG1655 Strr ⌬rbsK::cam has a 759-bp CACAGGT-3 . For E. coli MG1655 rbsK PCR amplification, the primers were Ј Ј Ј deletion of the 929-bp rbsK gene (7). It is unable to convert ribose to ribose-5- 5 -GGTTGTATGACCTGATGGTGAC-3 and 5 -GAGAAACTG TTGAGGT Ј phosphate, lacks ribokinase, and is both streptomycin and chloramphenicol re- AGAAACG-3 . The same primers were used for sequencing. For E. coli Nissle

⌬ Ј on October 11, 2018 by guest sistant. It is referred to in the text as E. coli MG1655 ⌬rbsK. E. coli MG1655 Strr 1917 rbsK PCR amplification, the primers were 5 -CGTTGTATGACCTGAT Ј Ј Ј ⌬fucAO ⌬rbsK::cam has the identical 1,542-bp deletion in the fucA and fucO GGTGAC-3 and 5 -GAGAAACT GTTGAGGTAGAAACG-3 . The same genes as described above but is missing the kanamycin cassette. In addition, it has primers were used for sequencing. a 759-bp deletion in the rbsK gene as described above. It is both streptomycin Mouse colonization experiments. The method used to compare the large resistant and chloramphenicol resistant and is referred to in the text as E. coli intestine-colonizing abilities of E. coli strains in mice has been described previ- MG1655 ⌬fucAO ⌬rbsK. E. coli MG1655 Strr ⌬fucK::kan has a 993-bp deletion ously (52, 53, 55). Briefly, three male CD-1 mice (5 to 8 weeks old) were given of the 1,448-bp fucK gene. It is unable to convert fuculose to fuculose-1-phos- drinking water containing streptomycin sulfate (5 g/liter) for 24 h to eliminate phate, lacks L-fuculokinase, and is both streptomycin and kanamycin resistant. resident facultative bacteria (36). Following 18 h of starvation for food and water, Wild-type E. coli Nissle 1917 was obtained from Dean Hamer of the National the mice were fed 1 ml of 20% (wt/vol) containing LB-grown E. coli Cancer Institute. E. coli Nissle 1917 Strr is a spontaneous streptomycin-resistant strains, as described in the Results section. After ingesting the bacterial suspen- mutant of the wild-type strain. E. coli Nissle 1917 Strr Nalr is a spontaneous sion, both the food (Harlan Teklad Mouse and Rat Diet, Madison, WI) and nalidixic acid-resistant mutant of E. coli Nissle 1917 Strr and is referred to in the streptomycin-water were returned to the mice, and1goffeces was collected text as E. coli Nissle 1917. The following deletion mutants were constructed from after 5 h, 24 h, and on odd-numbered days at the indicated times. Mice were E. coli Nissle 1917 Strr as described below (see the section on mutant construc- housed individually in cages without bedding and were placed in clean cages tion): E. coli Nissle 1917 Strr ⌬fucAO::kan, E. coli Nissle 1917 Strr ⌬rbsK::kan, E. daily. Fecal samples (no older than 24 h) were homogenized in 1% Bacto coli Nissle 1917 Strr ⌬fucAO ⌬rbsK::cam, and E. coli Nissle 1917 Strr ⌬fucK::cam. tryptone, diluted in the same medium, and plated on MacConkey agar plates ␮ The mutants contain the same deletions as their E. coli MG1655 counterparts with appropriate antibiotics. Plates contained streptomycin sulfate (100 g/ml) ␮ ␮ and are referred to in the text as E. coli Nissle 1917 ⌬fucAO, E. coli Nissle 1917 and nalidixic acid (50 g/ml), streptomycin sulfate (100 g/ml) and kanamycin ␮ ␮ ⌬rbsK, E. coli Nissle 1917 ⌬fucAO ⌬rbsK, and E. coli Nissle 1917 ⌬fucK, respec- sulfate (40 g/ml), or streptomycin sulfate (100 g/ml) and chloramphenicol (30 ␮ tively. g/ml). Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO). All Media and growth conditions. Luria broth (LB) was made as described by plates were incubated for 18 to 24 h at 37°C prior to counting. Each colonization Revel (48). Luria agar is LB containing 12 g of Bacto agar (Difco) per liter. experiment was performed at least twice, with essentially identical results. Pooled MacConkey agar (Difco) was prepared according to the manufacturer’s instruc- data from at least two independent experiments are presented in the figures. tions. For testing the ability of E. coli strains to utilize D-ribose or L-fucose, overnight cultures grown in LB were washed twice in M9 minimal medium (no RESULTS carbon source), 10 ␮l of the washed cultures was transferred to 10 ml of M9 minimal medium (35) containing reagent-grade glycerol (0.4%, wt/wt) as the sole Sequencing and growth of mutants in vitro. All mutants carbon and energy source, and cultures were incubated at 37°C with shaking in 125-ml tissue culture bottles. Growth and the lack of growth were assessed used in this study were sequenced to be sure that the deletions visually. For testing the ability of L-fucose to induce the utilization of D-ribose in were in the expected places in the chromosome (see Materials E. coli MG1655 and in E. coli Nissle 1917 ⌬fucAO and ⌬fucK mutants, the and Methods). In addition, each mutant was tested for growth mutants were grown twice in M9 minimal medium containing glycerol (0.2%, in M9 minimal medium containing 0.4% (wt/wt) glycerol, fu- A wt/wt) as described above and then were washed and resuspended at an 600 of cose, or ribose as the sole carbon and energy source. All mu- 0.1 in three separate 20-ml volumes of M9 minimal medium containing either 0.05% fucose, 0.15% ribose, or 0.05% fucose and 0.15% ribose. The cultures tants grew normally with glycerol as the sole carbon and energy were incubated at 37°C with shaking in 125-ml tissue culture bottles. Growth was source. Furthermore, all mutants with a ⌬fucAO deletion or a VOL. 75, 2007 L-FUCOSE AND E. COLI COLONIZATION 5467 Downloaded from

FIG. 1. Fucose operons and degradation pathway (A) and the ribose operon and degradation pathway (B). Arrows above genes indicate promoters and the direction of transcription. MFS, major facilitator superfamily; ABC, ATP-binding cassette.

⌬fucK deletion failed to grow with fucose as the sole carbon colonizer than expected, essentially being eliminated by day 15 and energy source, and those with a ⌬rbsK deletion failed to after feeding (Fig. 2C). These data suggested the possibility http://iai.asm.org/ grow with ribose as the sole carbon and energy source. More- that the E. coli MG1655 ⌬fucAO mutant switched to ribose in over, ⌬fucAO ⌬rbsK double mutants failed to grow on a mix- the intestine. If true, the E. coli MG1655 ⌬fucAO ⌬rbsK double ture of fucose and ribose. mutant should be a far worse colonizer than the E. coli Mouse intestinal colonization of E. coli MG1655 ⌬fucAO MG1655 ⌬fucAO mutant, despite differing only in the ability to and ⌬rbsK mutants. Unlike its wild-type parent, E. coli utilize ribose for growth. Indeed, the E. coli MG1655 ⌬fucAO MG1655 ⌬fucAO cannot convert fuculose-1-phosphate to di- ⌬rbsK double mutant was rapidly eliminated in competition hydroxyacetone phosphate and lactaldehyde (Fig. 1A) and with E. coli MG1655 ⌬fucAO (Fig. 2D), suggesting that the E.

therefore is unable to use fucose as a carbon and energy coli MG1655 ⌬fucAO mutant, in contrast to the wild-type E. on October 11, 2018 by guest source. Similarly, E. coli MG1655 ⌬rbsK cannot phosphorylate coli MG1655, utilizes ribose for growth in the intestine. ribose to ribose-5-phosphate and therefore is unable to utilize When mice are fed high numbers (1010 CFU/mouse) of a ribose as a carbon and energy source (Fig. 1B). When mice wild-type E. coli strain (resistant to streptomycin) and low were fed 105 CFU each of wild-type E. coli MG1655 and E. coli numbers (105 CFU/mouse) of the same wild-type strain (e.g., MG1655 ⌬fucAO, the wild-type E. coli MG1655 grew to a level resistant to streptomycin and nalidixic acid), they maintain the of about 108 CFU/g of feces within 1 day after feeding and then initial ratio of their input values (52), as would be expected of within a few days stabilized at a level of about 107 CFU/g of two strains that use all nutrients equally well. It then would be feces, whereas E. coli MG1655 ⌬fucAO initially grew to a level expected that if wild-type E. coli MG1655 was fed to mice in of only about 107 CFU/g of feces and within a few days stabi- high numbers (1010 CFU/mouse) and the E. coli MG1655 lized at a level of about 105 CFU/g of feces (Fig. 2A). When ⌬fucAO mutant was fed to the same mice in low numbers (105 mice were fed 105 CFU each of wild-type E. coli MG1655 and CFU/mouse) and the only difference between the two strains E. coli MG1655 ⌬rbsK, both grew at the same rate in the was their ability to utilize fucose for growth, the ⌬fucAO mu- intestine to about 108 CFU/g of feces, and only thereafter did tant would stabilize in numbers 5 orders of magnitude lower the E. coli MG1655 wild type, which stabilized at a level of than those for both strains being fed to mice in equal numbers, about 107 CFU/g of feces, develop a very slight advantage, at i.e., a total of about 7 orders of magnitude. On the other hand, most fourfold, over E. coli MG1655 ⌬rbsK (Fig. 2B). There- if the E. coli MG1655 ⌬fucAO mutant was using ribose and the fore, E. coli MG1655 appeared to use fucose during the wild-type E. coli MG1655 was not, the E. coli MG1655 ⌬fucAO initiation stage of the colonization process but appeared to mutant could conceivably grow from low numbers to higher stop using it during maintenance. In contrast, ribose did not numbers in the presence of high numbers of the wild-type appear to be used to any great extent during either stage of strain and could colonize at the level observed when both colonization. strains were fed to the mice in low numbers, i.e., only 2 orders E. coli MG1655 ⌬fucAO appears to switch to ribose in the of magnitude lower than the level of the wild type (Fig. 1A). intestine. Since E. coli MG1655 ⌬rbsK was nearly as good a This is precisely what happened, i.e., when mice were fed 1010 colonizer as wild-type E. coli MG1655, it was expected that the CFU of the wild-type E. coli MG1655 and 105 CFU of the E. colonizing ability of an E. coli MG1655 ⌬fucAO ⌬rbsK double coli MG1655 ⌬fucAO mutant, the mutant grew from a level of mutant would closely mimic the colonizing ability of E. coli 5 orders of magnitude lower than that of its parent to only 2 MG1655 ⌬fucAO. Surprisingly, when mice were fed 105 CFU orders of magnitude lower within a few days (Fig. 2E). each of the wild-type E. coli MG1655 and E. coli MG1655 The switch to ribose appears to require fuculose-1-phos- ⌬fucAO ⌬rbsK, the double mutant proved to be a far worse phate. Because the E. coli MG1655 ⌬fucAO mutant cannot 5468 AUTIERI ET AL. INFECT.IMMUN. Downloaded from http://iai.asm.org/ on October 11, 2018 by guest

FIG. 2. Colonization of the mouse intestine by E. coli MG1655 ⌬fucAO, E. coli MG1655 ⌬rbsK, and E. coli MG1655 ⌬fucAO ⌬rbsK. Sets of three mice were fed 105 CFU of E. coli MG1655 (f) and 105 CFU of MG1655 ⌬fucAO (E) (A); 105 CFU of E. coli MG1655 (f) and 105 CFU of E. coli MG1655 ⌬rbsK (E) (B); 105 CFU of E. coli MG1655 (f) and 105 CFU of E. coli MG1655 ⌬fucAO ⌬rbsK (E) (C); 105 CFU of E. coli MG1655 ⌬fucAO (f) and 105 CFU of E. coli MG1655 ⌬fucAO ⌬rbsK (E) (D); and 1010 CFU of E. coli MG1655 (f) and 105 CFU of E. coli MG1655 ⌬fucAO (E) (E). At the indicated times, fecal samples were homogenized, diluted, and plated as described in Materials and Methods.

Bars representing standard errors of the log10 means of CFU per gram of feces for six mice are presented for each time point except for panels A and B, for which data from 12 mice are presented. Each colonization curve has the specific strain genotype immediately above or below it. wt, wild-type E. coli MG1655. convert fuculose-1-phosphate to dihydroxyacetone phosphate phate in the presence of fucose. Since the E. coli MG1655 and lactaldehyde (8) and because fuculose-1-phosphate is re- ⌬fucAO mutant appeared to be using ribose for growth in the quired for induction of the fucPIKUR operon (4), E. coli intestine, the question was whether the switch to ribose re- MG1655 ⌬fucAO most likely accumulates fuculose-1-phos- quires the presence of fuculose-1-phosphate. An E. coli VOL. 75, 2007 L-FUCOSE AND E. COLI COLONIZATION 5469 Downloaded from

FIG. 3. Colonization of the mouse intestine by E. coli MG1655 ⌬fucK. Sets of three mice were fed 105 CFU of E. coli MG1655 (f) and 105 CFU of MG1655 ⌬fucK (E) (A) or 1010 CFU of E. coli MG1655 (f) and 105 CFU of E. coli MG1655 ⌬fucK (E) (B). At the indicated times, fecal samples were homogenized, diluted, and plated as described in Materials and Methods. Bars representing standard errors of the log10 means of CFU per gram of feces for 12 mice are presented for each time point in panel A and for 6 mice in panel B. Each colonization curve has the specific strain genotype immediately above or below it. wt, wild-type E. coli MG1655. http://iai.asm.org/ MG1655 ⌬fucK mutant, which is defective in the phosphory- or absence of fucose (data not shown). Collectively, these in lation of fuculose to fuculose-1-phosphate (Fig. 1A), was em- vitro data support the idea that it is accumulation of fuculose- ployed to address this question. When mice were fed 105 CFU 1-phosphate that stimulates growth of E. coli MG1655 ⌬fucAO each of wild-type E. coli MG1655 and E. coli MG1655 ⌬fucK, on ribose in the mouse intestine. the mutant colonized at most fivefold below the level of its Fucose has no effect on utilization of N-acetylglucosamine, wild-type parent (Fig. 3A). When the E. coli MG1655 ⌬fucK but it inhibits the utilization of and for mutant was fed to mice at 105 CFU/mouse and wild-type E. coli growth by E. coli MG1655 ⌬fucAO in vitro. Both E. coli 10

MG1655 was simultaneously fed at 10 CFU/mouse, the E. MG1655 ⌬fucK and E. coli MG1655 ⌬fucAO are unable to use on October 11, 2018 by guest coli MG1655 ⌬fucK mutant was eliminated (Fig. 3B). There- fucose for growth, but E. coli MG1655 ⌬fucAO switches to fore, the E. coli MG1655 ⌬fucK mutant does not use ribose, ribose in the intestine, whereas the switch to ribose is not made and neither does it switch to any other nutrient in the intestine by E. coli MG1655 ⌬fucK. Why then is E. coli MG1655 ⌬fucK to allow it to grow up in the presence of high numbers of a better colonizer than E. coli MG1655 ⌬fucAO when each is wild-type E. coli MG1655. Moreover, since the only difference in competition with wild-type E. coli MG1655 (compare Fig. between the E. coli MG1655 ⌬fucK mutant and the E. coli 2A to 3A)? One possibility was that while accumulation of MG1655 ⌬fucAO mutant is the inability of the E. coli MG1655 fuculose-1-phosphate in E. coli MG1655 ⌬fucAO stimulates ⌬fucK mutant to accumulate fuculose-1-phosphate, i.e., nei- utilization of ribose, it might simultaneously inhibit utilization ther mutant is able to make dihydroxyacetone phosphate and of other sugars. Growth experiments analogous to those de- lactaldehyde (Fig. 1A), it would appear that the switch of E. scribed for the effect of fucose on ribose utilization also were coli MG1655 ⌬fucAO to ribose in the intestine requires the performed to determine the effect of fucose on utilization of accumulation of fuculose-1-phosphate. N-acetylglucosamine, galactose, and mannose for growth by E. Fucose stimulates the utilization of ribose for growth by E. coli MG1655 ⌬fucAO and E. coli MG1655 ⌬fucK in vitro. coli MG1655 ⌬fucAO in vitro but not for growth by E. coli Indeed, although 0.05% fucose had no effect on the utilization MG1655 ⌬fucK. Since it appeared that fucose stimulates utili- of 0.15% N-acetylglucosamine by E. coli MG1655 ⌬fucAO in zation of ribose by E. coli MG1655 ⌬fucAO in the intestine, vitro (Fig. 5A), it greatly reduced the utilization of both galac- growth experiments were performed to determine whether fu- tose and mannose (Fig. 5B and C). In contrast, utilization of cose could stimulate utilization of ribose by E. coli MG1655 N-acetylglucosamine, galactose, and mannose was unaltered by ⌬fucAO in vitro. E. coli MG1655 ⌬fucAO and E. coli MG1655 fucose in the E. coli MG1655 ⌬fucK mutant (data not shown). ⌬fucK were grown in M9 minimal medium in the presence It therefore seems likely that E. coli MG1655 ⌬fucAO is a of 0.05% fucose, 0.15% ribose, and a mixture of 0.05% worse colonizer than E. coli MG1655 ⌬fucK, despite using fucose and 0.15% ribose. As expected, neither strain grew ribose more efficiently, because accumulation of fuculose-1- with fucose as the sole carbon and energy source (Fig. 4). phosphate inhibits its ability to compete for other sugars that However, fucose stimulated more rapid growth of E. coli are normally used for growth in the intestine. MG1655 ⌬fucAO on ribose (Fig. 4A) but had no effect on the Fucose stimulates ribose utilization for growth by wild-type rate of growth of E. coli MG1655 ⌬fucK on ribose (Fig. 4B). E. coli MG1655 in vitro. In vitro growth experiments also were That the stimulation of ribose utilization required a functional performed to determine whether fucose could stimulate utili- rbsK gene was shown by the fact that the E. coli MG1655 zation of ribose by wild-type E. coli MG1655 in vitro. In this ⌬fucAO ⌬rbsK mutant failed to grow on ribose in the presence case, it was necessary to use a concentration of fucose that 5470 AUTIERI ET AL. INFECT.IMMUN. Downloaded from

FIG. 4. Growth of E. coli MG1655 ⌬fucAO (A) and E. coli MG1655 ⌬fucK (B) in M9 minimal medium in the presence of 0.05% (wt/wt) fucose (f), 0.15% (wt/wt) ribose (}), or a mixture of 0.05% (wt/wt) fucose and 0.15% (wt/wt) ribose (E). E. coli MG1655 ⌬fucAO and E. coli MG1655 ⌬fucK were grown in M9 minimal medium containing glycerol (0.2%, wt/wt), washed, and resuspended in M9 minimal medium containing the appropriate sugars (see Materials and Methods). Incubation was at 37°C with aeration. A600 readings at the indicated times are presented. Growth experiments were performed at least three times. The results of typical experiments are shown. http://iai.asm.org/ would not allow E. coli MG1655 to grow but might still stim- Mice also were fed 105 CFU each of wild-type E. coli Nissle ulate ribose utilization. Therefore, E. coli MG1655 was grown 1917 and an E. coli Nissle 1917 ⌬fucK mutant. The E. coli in the presence of 0.005% fucose, 0.15% ribose, or a mixture of Nissle 1917 ⌬fucK mutant colonized at a level of between 1 and 0.005% fucose and 0.15% ribose. Wild-type E. coli MG1655 2 orders of magnitude lower than that of the wild-type strain did not grow in the presence of 0.005% fucose, but 0.005% (Table 1). In addition, when mice were fed 1010 CFU of wild- fucose did stimulate the utilization of ribose by wild-type E. type E. coli Nissle 1917 and 105 CFU of E. coli Nissle 1917 coli MG1655 (Fig. 6). It therefore appears that stimulation of ⌬fucK, the E. coli Nissle 1917 ⌬fucK mutant was essentially

ribose utilization by fucose also can occur in wild-type E. coli eliminated (Table 1). Collectively, these data suggest that the on October 11, 2018 by guest MG1655 at fucose concentrations too low to allow growth, E. coli Nissle 1917 ⌬fucK mutant does not switch to ribose in suggesting the possibility that under the right conditions, fu- the intestine and that, like the E. coli MG1655 ⌬fucAO mutant, cose could signal wild-type E. coli MG1655 to utilize ribose in the E. coli Nissle 1917 ⌬fucAO mutant switches to ribose for the intestine. growth in the intestine, mediated by the accumulation of fu- E. coli Nissle 1917 ⌬fucAO also switches to ribose in the culose-1-phosphate. intestine. Experiments were conducted to determine whether Fucose stimulates the utilization of ribose by wild-type E. the switch to ribose in the intestine by E. coli MG1655 ⌬fucAO coli Nissle 1917 and by E. coli Nissle 1917 ⌬fucAO for growth was an isolated case or whether other E. coli strains carry out in vitro. Growth experiments were performed to determine the switch. E. coli Nissle 1917 is a commensal strain that has whether fucose could stimulate utilization of ribose in the E. been used successfully as a probiotic agent to treat gastroin- coli Nissle 1917 ⌬fucAO mutant in vitro. The E. coli Nissle testinal infections in humans since the early 1920s (50). Like E. 1917 ⌬fucAO mutant and the E. coli Nissle 1917 ⌬fucK mutant coli MG1655 ⌬fucAO,anE. coli Nissle 1917 ⌬fucAO mutant were grown in vitro in the presence of 0.05% fucose, 0.15% colonized at a level about 2 orders of magnitude less than that ribose, and 0.05% fucose plus 0.15% ribose. As expected, nei- of its wild-type parent when 105 CFU of each strain was fed to ther strain grew on fucose as the sole carbon and energy mice (Table 1). Also like E. coli MG1655 ⌬rbsK,anE. coli source. However, fucose was able to induce more rapid growth Nissle 1917 ⌬rbsK mutant colonized at a level of only about of E. coli Nissle 1917 ⌬fucAO on ribose (Fig. 7A). Fucose did fourfold lower than that of its wild-type parent when 105 CFU not induce more rapid growth of the E. coli Nissle 1917 ⌬fucK of each strain was fed to mice (Table 1). In addition, an E. coli mutant on ribose (Fig. 7B). Moreover, fucose had no effect on Nissle 1917 ⌬fucAO ⌬rbsK mutant colonized at a level of about N-acetylglucosamine utilization but inhibited both mannose 5 orders of magnitude lower than that of its wild-type parent and galactose utilization in the E. coli Nissle 1917 ⌬fucAO when 105 CFU of each strain was fed to mice, i.e., greater than mutant (data not shown). Fucose had no effect on N-acetyl- 2 orders of magnitude lower than expected (Table 1). Further- glucosamine, galactose, or mannose utilization by E. coli Nissle more, when mice were fed 1010 CFU of wild-type E. coli Nissle 1917 ⌬fucK (data not shown). It therefore appears that as in 1917 and 105 CFU of the E. coli Nissle 1917 ⌬fucAO mutant, the case of E. coli MG1655, accumulation of fuculose-1-phos- the mutant grew from a level of 5 orders of magnitude lower phate induces more rapid growth of the commensal E. coli than that of its parent to between 1.5 and 2 orders of magni- Nissle 1917 ⌬fucAO mutant on ribose in vitro, has no effect on tude lower within a few days (Table 1). Therefore, like E. coli N-acetylglucosamine utilization, and inhibits both galactose MG1655 ⌬fucAO, the E. coli Nissle 1917 ⌬fucAO mutant ap- and mannose utilization. Finally, wild-type E. coli Nissle 1917 pears to use ribose for growth in the intestine. was grown in the presence of 0.005% fucose, 0.15% ribose, or VOL. 75, 2007 L-FUCOSE AND E. COLI COLONIZATION 5471 Downloaded from http://iai.asm.org/ on October 11, 2018 by guest

FIG. 5. Growth of E. coli MG1655 ⌬fucAO in M9 minimal medium in the presence of 0.05% (wt/wt) fucose (f), 0.15% (wt/wt) N- acetylglucosamine (}), or a mixture of 0.05% (wt/wt) fucose and 0.15% (wt/wt) N-acetylglucosamine (E) (A); 0.05% (wt/wt) fucose (f), 0.15% (wt/wt) mannose (}), or a mixture of 0.05% (wt/wt) fucose and 0.15% (wt/wt) mannose (E) (B); and 0.05% (wt/wt) fucose (f), 0.15% (wt/wt) galactose (}), or a mixture of 0.05% (wt/wt) fucose and 0.15% (wt/wt) galactose (E) (C). E. coli MG1655 ⌬fucAO was grown in M9 minimal medium containing glycerol (0.2%, wt/wt), washed, and resuspended in M9 minimal medium containing the appropriate sugars (see Materials and

Methods). Incubation was at 37°C with aeration. A600 readings at the indicated times are presented. Growth experiments were performed at least three times. The results of typical experiments are shown. a mixture of 0.005% fucose and 0.15% ribose. Again, like E. shown that when E. coli was grown in a chemostat under coli MG1655, the wild-type E. coli Nissle 1917 did not grow in -limiting conditions, genes associated with the the presence of 0.005% fucose, but 0.005% fucose did stimu- and galactose operons were up-regulated relative to batch cul- late its use of ribose (Fig. 7C). tures grown under conditions in which glucose was not limited (18). Similarly, with acetate as the sole carbon source, genes DISCUSSION encoding transporters for galactose, ribose, and N-acetylglu- cosamine were up-regulated relative to growth with glucose It is becoming increasingly clear that in the face of compe- as the sole carbon source (44). Moreover, using Biolog AN tition from a complex microflora, E. coli simultaneously uses several, presumably limiting, sugars for growth in the mouse MicroPlate respiration analysis, it has recently been shown that when E. coli MG1655 was grown in a chemostat under glucose- intestine, including D-gluconate, N-acetyl-D-glucosamine, limiting conditions, despite the absence of inducers a wide D-glucuronate, and sialic acid (7, 10, 30, 31). The simultaneous use of several sugars for growth is not unprecedented. In fact, variety of catabolic functions were derepressed (26). E. coli has been shown to utilize D-glucose, D-galactose, D- There are two divergently transcribed operons for fucose maltose, D-ribose, L-, and D- simultaneously utilization in E. coli (Fig. 1A), the fucPIKUR operon, which in a chemostat under carbon-limiting conditions (32). It also takes fucose to fuculose-1-phosphate, and the fucAO operon, appears that E. coli is capable of preparing itself for the ap- which takes fuculose-1-phosphate to dihydroxyacetone phos- pearance of alternative carbon sources when growing under phate and lactaldehyde (8). Fuculose-1-phosphate is the effec- glucose-limiting conditions or when utilizing acetate as the sole tor of induction of both fucose operons through the FucR carbon source (18, 44). Thus, DNA microarray analysis has activator protein (4, 8, 9). In the present study, evidence is 5472 AUTIERI ET AL. INFECT.IMMUN.

presence of (27). It is therefore not farfetched to suggest the possibility that in commensal E. coli strains fuculose-1- phosphate and FucR also regulate more than one operon. It also is possible that fuculose-1-phosphate interacts with the RbsR repressor protein or an as-yet unidentified protein to relieve repression of the ribose operon. Finally, it is possible that fuculose-1-phosphate is involved in opening a gate for ribose entry into the cells via a preexisting RbsABC trans- porter or in allosterically activating more of the ribose gene products. It appears that accumulation of fuculose-1-phosphate inhib- its growth of both E. coli MG1655 ⌬fucAO and E. coli Nissle 1917 ⌬fucAO on mannose and galactose. This finding is not

unexpected; i.e., it is well known that intracellular accumula- Downloaded from tion of phosphates is inhibitory to E. coli growth, al- though the reasons remain unclear (6, 11, 13, 16, 17, 56). FIG. 6. Growth of wild-type E. coli MG1655 in M9 minimal me- However, in view of the fact that sugar phosphates normally dium in the presence of 0.005% (wt/wt) fucose (f), 0.15% (wt/wt) } inhibit growth, it is unexpected that accumulation of fuculose- ribose ( ), or a mixture of 0.005% (wt/wt) fucose and 0.15% (wt/wt) ⌬ ribose (E). Wild-type E. coli MG1655 was grown in M9 minimal 1-phosphate would stimulate growth of E. coli MG1655 fucAO medium containing glycerol (0.2%, wt/wt), washed, and resuspended in and E. coli Nissle 1917 ⌬fucAO on ribose. Nevertheless, stim- M9 minimal medium containing the appropriate sugars (see Materials ulation of ribose utilization by fuculose-1-phosphate appears and Methods). Incubation was at 37°C with aeration. A600 readings at to be a normal physiological process; i.e., fucose at a concen-

the indicated times are presented. Growth experiments were per- http://iai.asm.org/ formed at least three times. The results of typical experiments are tration too low to allow growth (0.005%) stimulated ribose shown. utilization in both wild-type E. coli MG1655 and wild-type E. coli Nissle 1917 in vitro (Fig. 6 and 7C). In this context, al- though both fucose operons are activated by FucR in the presented suggesting that accumulation of fuculose-1-phos- presence of fuculose-1-phosphate (4, 9), it is possible that at phate through induction of the fucPIKUR operon in two com- low fucose concentrations the fucPIKUR operon is more highly mensal strains, E. coli MG1655 ⌬fucAO and E. coli Nissle 1917 expressed than the fucAO operon in wild-type E. coli MG1655, ⌬fucAO, stimulates growth on ribose both in the intestine and which could result in the accumulation of fuculose-1-phos-

in vitro. However, in a fucAO mutant, in addition to accumu- phate. In fact, the two fucose operons can be expressed differ- on October 11, 2018 by guest lation of fuculose-1-phophate, it would be expected that the entially (45, 57). The finding that low levels of fucose signal the fuculose transporter (FucP) encoded by fucP, the FucR acti- wild-type strains to grow more rapidly on ribose raises the vator protein encoded by fucR, and the L-fucose mutarotase possibility that it also occurs in the intestine, perhaps in a encoded by fucU also would accumulate. It recently has been minor niche. Such wild-type subpopulations presumably would reported (49) that the L-fucose mutarotase can convert ␤-D- be at the level of the population size of the ⌬fucAO mutants in pyranoribose, the form transported into the cell, to ␣-D-ribo- the presence of the wild-type strains, i.e., 100-fold below the (49). ␣-D-Ribofuranose is required for ribose catab- olism (40). It also has recently been reported that D-fructose possibly can enter an E. coli fucA mutant via FucP (29). If TABLE 1. Mouse intestinal colonization of E. coli Nissle 1917 ribose were to enter ⌬fucAO cells via FucP, it then could mutants relative to that of wild-type E. coli Nissle 1917 induce the ribose operon. In this case and in the case of the Difference between log mutarotase, the fuculose-1-phosphate would be necessary 10 Defect in CFU of the wild type and Sugar Inputa only for inducing the fucPIKUR operon. Whether the L-fucose mutant log10 CFU mutant on day: permease and/or the L-fucose mutarotase is involved in stim- 39 ulation of ribose utilization is presently under investigation. Fucoseb ⌬fucAO 0.13 Ϯ 0.15 2.6 Ϯ 0.5 2.3 Ϯ 0.2 At the present time, it is not clear how or if fuculose-1- Riboseb ⌬rbsK 0.18 Ϯ 0.15 0.03 Ϯ 0.08 0.45 Ϯ 0.23 phosphate is involved either by itself or as an effector in com- Fucose, riboseb ⌬fucAO ⌬rbsK 0.27 Ϯ 0.33 2.6 Ϯ 0.4 4.7 Ϯ 0.4 Fucoseb ⌬fucK 0.22 Ϯ 0.18 0.60 Ϯ 0.15 1.3 Ϯ 0.1 bination with a regulatory protein in inducing the rbsDABCKR Fucosec ⌬fucAO 4.7 Ϯ 0.6 1.3 Ϯ 0.3 1.7 Ϯ 0.2 operon. It is possible that accumulated fuculose-1-phosphate Fucosec ⌬fucK 4.5 Ϯ 0.5 5.1 Ϯ 0.4 5.5 Ϯ 0.3 acts as an effector of FucR to directly activate or release re- a Input values represent the mean of the log10 CFU of the E. coli Nissle 1917 Ϯ pression of transcription of the rbsDABCKR operon. In Bac- mutant subtracted from the log10 CFU of the wild-type E. coli Nissle 1917 ( the teroides thetaiotaomicron, L-fucose has been implicated through log10 standard errors of the means) fed to six mice. b Mice were fed 105 CFU each of an E. coli Nissle 1917 mutant and its its FucR protein, which is unrelated to the E. coli FucR pro- wild-type parent. Mice were transferred to fresh cages every day, and feces no tein, to both induce the fucose operon and, along with FucR, older than 24 h were assayed every other day for 15 days. At each time point, for each mouse the log10 CFU/gram of feces for the mutant was subtracted from the to corepress an as-yet unidentified locus named cps (control of Ϯ log10 CFU/gram of feces for the wild type. The log10 mean of the difference the signal production) that may be responsible for inducing the log10 standard error of the mean of day 3 and day 9 fecal data from at least 6 mice mammalian host to make hydrolysable fucosylated are shown. c The same procedure as that described in footnote b was followed, except that (24). Moreover, it has been shown that transcription of the mice were fed 105 CFU of an E. coli Nissle 1917 mutant and 1010 CFU of its ribose operon is repressed by XylR, the xylose regulator, in the wild-type parent. VOL. 75, 2007 L-FUCOSE AND E. COLI COLONIZATION 5473 Downloaded from http://iai.asm.org/ on October 11, 2018 by guest

FIG. 7. Growth of E. coli Nissle 1917 ⌬fucAO in M9 minimal medium in the presence of 0.05% (wt/wt) fucose (f), 0.15% (wt/wt) ribose (}), or a mixture of 0.05% (wt/wt) fucose and 0.15% (wt/wt) ribose (E) (A); growth of E. coli Nissle 1917 ⌬fucK in M9 minimal medium in the presence of 0.05% (wt/wt) fucose (f), 0.15% (wt/wt) ribose (}), or a mixture of 0.05% (wt/wt) fucose and 0.15% (wt/wt) ribose (E) (B); and growth of wild-type E. coli Nissle 1917 in M9 minimal medium in the presence of 0.005% (wt/wt) fucose (f), 0.15% (wt/wt) ribose (}), or a mixture of 0.005% (wt/wt) fucose and 0.15% (wt/wt) ribose (E) (C). Strains were grown in M9 minimal medium containing glycerol (0.2%, wt/wt), washed, and resuspended in M9 minimal medium containing the appropriate sugars (see Materials and Methods). Incubation was at 37°C with aeration. A600 readings at the indicated times are presented. Growth experiments were performed at least three times. The results of typical experiments are shown. wild-type population (Fig. 2E), and would go unnoticed in regulation designed for E. coli and perhaps other members of routine colonization experiments. Experiments designed to the microflora to more efficiently utilize the available limiting test this possibility are currently in progress. multiple nutrients to maintain growth rates sufficient to avoid The finding that E. coli MG1655 ⌬fucAO and E. coli Nissle washout from the intestine. 1917 ⌬fucAO mutants switch to ribose in the intestine because they accumulate fuculose-1-phosphate not only supports the ACKNOWLEDGMENTS nutrient-niche theory but also has far-reaching implications This research was supported by Public Health Service grant AI with respect to the stability of the commensal flora in the 48945 to T.C. and P.S.C. intestine. E. coli and presumably other members of the intes- We are grateful to Shelley Brown for excellent technical assistance. tinal microflora use several limiting nutrients simultaneously REFERENCES for growth (7, 10, 30, 31, 37), but how any member of the 1. Allan, A. 1981. Structure and function of gastrointestinal mucus, p. 637–639. microflora chooses the specific nutrients to use at any one time In L. R. Johnson (ed.), Physiology of the gastrointestinal tract. Raven Press, among those available to it is largely unknown. The data pre- New York, NY. sented here suggest that the intracellular pool size of a meta- 2. Anderson, J. D., W. A. Gillespie, and M. H. Richmond. 1973. Chemotherapy and antibiotic-resistance transfer between enterobacteria in the human gas- bolic intermediate involved in the metabolism of one nutrient tro-intestinal tract. J. Med. Microbiol. 6:461–473. plays a role in this process by signaling E. coli to rapidly switch 3. Atuma, C., V. Strugala, A. Allen, and L. Holm. 2001. The adherent gastro- intestinal mucus gel layer: thickness and physical state in vivo. Am. J. Physiol. to a second unrelated nutrient for growth. Thus, it is possible Gastrointest. Liver Physiol. 280:G922–G929. that a variety of metabolic intermediates comprise a tier of 4. Bartkus, J. M., and R. P. Mortlock. 1986. Isolation of a mutation resulting 5474 AUTIERI ET AL. INFECT.IMMUN.

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